Chitosan oligosaccharide chromatographic separation medium and its preparation method and application

Abstract

The present invention relates to a chitosan oligosaccharide chromatographic separation medium and a preparation method and application thereof. Styrene, divinylbenzene, sodium chloride, polyvinyl alcohol and n-butyl ether are dissolved in water and stirred to obtain a mixture A; a methylene blue aqueous solution is added to the mixture A to obtain a mixture B; an initiator is added to the mixture B to obtain a mixture C; the mixture C is subjected to a program heating and cooling reaction to obtain a polymer ball D having a core-shell structure; the polymer ball D is sulfonated to obtain a core-shell type strongly acidic ion exchange resin E; an acyl chloride reagent is added for reflux reaction to obtain a core-shell type sulfonyl chloride resin F; the core-shell type sulfonyl chloride resin F is added with aminocarboxylic acid for reflux reaction to obtain a chitosan oligosaccharide chromatographic separation medium. The material of the present invention is simple to prepare, the functional group is stable and not easy to lose, the separation efficiency is high, the one-time sample processing amount of the medium per unit volume is large, the cost is lower than that of the existing method, and it has important application value for the separation, purification and industrial production of chitosan oligosaccharides with a single degree of polymerization.

Description

Technical Field

[0001] The invention belongs to the technical field of chromatographic separation, and in particular relates to a chitosan oligosaccharide chromatographic separation medium and a preparation method and application thereof. Background Art

[0002] Ion exchange resins have the advantages of large load, fast flow rate and high resolution, and are suitable for the industrial separation of charged substances.

[0003] Currently, there are reported methods for separating chitosan oligosaccharides with a single degree of polymerization, such as CN201210146042.7, which uses imported dextran gel matrix ion exchange chromatography medium to separate chitosan oligosaccharides with a single degree of polymerization. The chromatography medium is expensive, and the one-time sample processing capacity is only at the gram level, which is not suitable for large-scale expansion and industrial application.

[0004] Traditional synthetic ion exchange resins often have problems such as long equilibrium time, severe product tailing, and low separation efficiency for charged substances with larger molecular weight.

[0005] The ion exchange medium synthesized at present is mainly a polymer sphere obtained by a single heating synthesis and then modified to prepare an ion exchange resin. This type of structure has uniform pore distribution, a longer mass transfer distance at the same particle size, a greater diffusion resistance in the pores, a longer equilibrium time for chitosan oligosaccharides with different polymerization degrees, and low separation efficiency.

[0006] The chromatographic media currently reported for efficient separation of chitosan oligosaccharides, such as CN106076298A, are prepared by grafting modification on a silica gel matrix, have a small one-time sample loading capacity, are analytical grade, and are limited to high performance liquid chromatography equipment. Although they have a good separation effect on chitosan oligosaccharides with a degree of polymerization of 2-6, the chromatographic medium loading capacity is low, and the one-time sample processing volume is only at the milligram level, which limits the large-scale production and high-value application of chitosan oligosaccharides with a single degree of polymerization. Summary of the invention

[0007] In order to solve the problem of low loading capacity or low separation efficiency of chromatographic media for efficient separation of chitosan oligosaccharides in the prior art, the present invention provides a chitosan oligosaccharide chromatographic separation medium and a preparation method and application thereof.

[0008] The purpose of the present invention can be achieved by the following technical solutions:

[0009] The invention provides a method for preparing a chitosan oligosaccharide chromatographic separation medium, comprising the following steps.

[0010] (1) dissolving styrene, divinylbenzene, sodium chloride, polyvinyl alcohol and n-butyl ether in water in a reaction kettle and stirring to obtain a mixture A;

[0011] (2) adding a methylene blue aqueous solution to the mixture A, stirring and mixing to obtain a mixture B;

[0012] (3) adding an initiator to mixture B and stirring to obtain mixture C;

[0013] (4) The mixture C is placed in a reaction kettle, and the temperature is raised and lowered programmatically under stirring conditions to react, and the product is separated by filtration, and ethanol is refluxed to obtain polymer spheres D having a core-shell structure;

[0014] (5) the polymer sphere D is swelled with chloroform and sulfonated to obtain a core-shell type strongly acidic ion exchange resin E;

[0015] (6) Swelling the core-shell type strong acid ion exchange resin E with chloroform, adding an acyl chloride reagent for reflux reaction, washing with dichloromethane and drying to obtain a core-shell type sulfonyl chloride resin F;

[0016] (7) The core-shell type sulfonyl chloride resin F is swollen with chloroform, dichloromethane is added as a reaction solvent, aminocarboxylic acid is added for reflux reaction, filtered and washed, and chitosan oligosaccharide chromatography separation medium is obtained.

[0017] In some embodiments of the present invention, in step (1), in the mixture A, the mass ratio of water, styrene, divinylbenzene, sodium chloride, polyvinyl alcohol and n-butyl ether is 300-500:100-200:5-50:5:0.1:0.5:5-50.

[0018] In some embodiments of the present invention, in step (2), the mass concentration of the methylene blue aqueous solution is 0.05-2%.

[0019] In some embodiments of the present invention, in step (2), the relationship between the amount of the methylene blue aqueous solution added and the mixture A is 0.2 ml: 400-800 g.

[0020] In some embodiments of the present invention, in step (3), the initiator is selected from one or more organic peroxides such as benzoyl peroxide, tert-butyl benzoyl peroxide, methyl ethyl ketone peroxide, etc., preferably benzoyl peroxide.

[0021] In some embodiments of the present invention, in step (3), the mass proportion of the initiator in the mixture C after addition is 0.1-0.5%.

[0022] In some embodiments of the present invention, in step (3), the stirring time is 1-3 hours, and the stirring speed is 100-400 r / min.

[0023] In some embodiments of the present invention, in step (4), the temperature is programmed to rise and fall at 45-95° C. under stirring conditions for 6-18 hours.

[0024] In some embodiments of the present invention, in step (4), after ethanol refluxes, the ethanol is washed with water and dried to obtain polymer spheres D.

[0025] In some embodiments of the present invention, in step (5), the polymer spheres D are swollen with chloroform for 4-24 hours, and the swelling volume ratio is 1:1-1:5.

[0026] In some embodiments of the present invention, in step (5), the sulfonation conditions are: adding sulfuric acid for sulfonation, the volume ratio of the sulfuric acid to the solution swollen with chloroform is 1:2-1:5, and the sulfonation temperature is 70-105°C.

[0027] In some embodiments of the present invention, in step (6), the chlorination agent is thionyl chloride, phosphorus trichloride or sulfonyl chloride.

[0028] In some embodiments of the present invention, in step (6), the reflux reaction conditions are: reflux at 45-65° C. for 4-10 h.

[0029] In some embodiments of the present invention, in step (7), the aminocarboxylic acid is selected from one or more aminocarboxylic acids such as α-aminoacetic acid, α-alanine or α-aminoglutaric acid.

[0030] In some embodiments of the present invention, in step (7), the reflux reaction conditions are: reflux at 45°C-60°C for 4-8h.

[0031] In the above method for preparing the chitosan oligosaccharide chromatographic separation medium of the present invention, the particle size and pore size distribution of the chitosan oligosaccharide chromatographic separation medium can be changed by adjusting the raw material ratio, stirring rate and reaction temperature.

[0032] The present invention further provides a chitosan oligosaccharide chromatographic separation medium obtained based on the preparation method.

[0033] The present invention further provides application of the chitosan oligosaccharide chromatographic separation medium obtained based on the preparation method in separation and purification of chitosan oligosaccharides.

[0034] In some embodiments of the present invention, the chitosan oligosaccharide chromatography separation medium is used for the separation of chitosan oligosaccharides with a single degree of polymerization.

[0035] In some embodiments of the present invention, when the chitosan oligosaccharide chromatography separation medium is used in the separation and purification of chitosan oligosaccharides, the stationary phase used is the chitosan oligosaccharide chromatography separation medium, and the chitosan oligosaccharide chromatography separation medium is an organic polymer skeleton chromatography medium with sulfonic acid group, carboxylic acid group and modified sulfonic acid and carboxylic acid as main functional groups.

[0036] In some embodiments of the present invention, when the chitosan oligosaccharide chromatography separation medium is used in the separation and purification of chitosan oligosaccharides, it can be used for the chromatographic separation of chitosan oligosaccharide mixtures with various polymerization degree composition ratios.

[0037] In some embodiments of the present invention, when the chitosan oligosaccharide chromatographic separation medium is used in the separation and purification of chitosan oligosaccharides, the chromatographic separation performed by the chromatographic column includes real moving bed chromatography, simulated moving bed chromatography, intermittent chromatographic column or fixed bed chromatography.

[0038] In some embodiments of the present invention, when the chitosan oligosaccharide chromatography separation medium is used in the separation and purification of chitosan oligosaccharides, the mobile phase used is a buffer system, a salt solution system or an acid-base solution system adjusted with inorganic acid, organic acid, inorganic base and organic base solution.

[0039] In some embodiments of the present invention, when the chitosan oligosaccharide chromatography separation medium is used in the separation and purification of chitosan oligosaccharides, a buffer solution, a salt solution or an acidic alkaline solution system is used as the mobile phase, and the separation of chitosan oligosaccharides with different polymerization degrees is achieved by adjusting the pH or the concentration of the elution system.

[0040] In some embodiments of the present invention, when the chitosan oligosaccharide chromatography separation medium is used in the separation and purification of chitosan oligosaccharides, the chromatography operating temperature range is 0-100°C.

[0041] In some embodiments of the present invention, when the chitosan oligosaccharide chromatographic separation medium is used for the separation of chitosan oligosaccharides with a single degree of polymerization, the method comprises the following steps:

[0042] (1) The chitosan oligosaccharide chromatographic separation medium is loaded into a chromatographic column as a chromatographic filler, the chitosan oligosaccharide loading concentration is 1%-99% (w / w), and the loading amount is 0.05BV-5BV;

[0043] (2) Then rinse with deionized water at 0.1-1 BV / min for 1-10 BV;

[0044] (3) Finally, the chitosan oligosaccharide mixture is eluted with a mobile phase at 0-100°C at a concentration of 0.001‰-60%, and the chitosan oligosaccharides with different single polymerization degrees are respectively combined.

[0045] The invention introduces multiple functional groups to weaken the interaction between the sulfonic acid group on the ion exchange resin and the chitosan oligosaccharide, thereby improving the tailing of the product under the condition of maintaining the separation degree of the chromatographic medium. At the same time, the introduction of multiple carboxyl functional groups can ensure the exchange capacity of the chromatographic medium. Compared with the existing chitosan oligosaccharide separation method, the modified core-shell ion exchange resin has higher mass transfer efficiency and higher exchange capacity, and reduces the tailing phenomenon while ensuring the separation accuracy.

[0046] The problem of long equilibrium time can be solved by reducing the mass transfer distance of ion exchange resin. Reducing the diffusion distance shortens the equilibrium time and improves the separation efficiency.

[0047] The chitosan oligosaccharide chromatographic separation medium provided by the present invention is a multifunctional cationic chromatographic medium, which can be used for the separation of chitosan oligosaccharides with a single degree of polymerization, and has higher mass transfer efficiency, lighter tailing phenomenon and higher separation degree compared with conventional cationic chromatographic separation media.

[0048] The present invention prepares and modifies a multilayer cation exchange resin, and applies it to the separation and purification of chitosan oligosaccharides with a single degree of polymerization, showing good separation performance. At the same time, the material is simple to prepare, the functional groups are stable and not easy to lose, the separation efficiency is high, the one-time sample processing volume per unit volume of medium is large, and the cost is lower than that of the existing method, and it has important application value for the separation and purification of chitosan oligosaccharides with a single degree of polymerization and industrial production.

[0049] Compared with the prior art, the present invention has the following beneficial effects:

[0050] The invention prepares a core-shell multifunctional ion exchange chromatography medium for separation of chitosan oligosaccharides, adopts programmed temperature rise and temperature drop, synthesizes a multilayer skeleton structure, and performs step-by-step sulfonation, thereby shortening the effective mass transfer distance of the chromatography medium and shortening the chitosan oligosaccharide adsorption equilibrium time. The introduction of multifunctional groups weakens the interaction strength between chitosan oligosaccharides and the chromatography medium, improves the tailing problem existing in the chromatographic separation of chitosan oligosaccharides, and has a higher separation efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 Infrared spectrum of synthetic resin of Example 1;

[0052] Figure 2 Example 1 Synthetic resin electron microscope image;

[0053] Figure 3 Infrared spectrum of synthetic resin of comparative example 1;

[0054] Figure 4 Infrared spectrum of synthetic resin of Example 2;

[0055] Figure 5 Example 4 Single polymerization degree chitosan oligosaccharide TLC detection chart;

[0056] Figure 6 Example 4 High performance liquid chromatography test results of single-degree-of-polymerization chitosan oligosaccharides. DETAILED DESCRIPTION

[0057] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0058] Example 1

[0059] 300g water, 100g styrene, 5g divinylbenzene, 5g sodium chloride, 0.1g polyvinyl alcohol, 5g n-butyl ether were stirred to obtain a mixture. Then 0.2ml of 0.05% methylene blue aqueous solution was added to the mixture. Then 2.0g benzoyl peroxide was added, the speed was 100r / min, and the mixture was stirred for 1h. After loading into the reactor, the mixture was reacted for 6h at 45°C and the speed was 100r / min. After filtering, the ethanol was refluxed, and the white spherical granular resin was washed and dried. The above 50g resin was taken, 150ml chloroform was added to swell for 4h, and then 100ml of 98% sulfuric acid was added to sulfonate at 70°C for 6h at a speed of 100r / min. After filtering, washing and drying, yellow spherical granular resin was obtained. Take 50g of the sulfonated resin, add 150ml chloroform to swell, and then add 150ml sulfonyl chloride reagent to reflux at 45°C for 4h, filter, wash with dichloromethane and dry. Take 50g of the dried resin, add 150ml of chloroform to swell, then add 100ml of dichloromethane, add 20g of α-aminoglutaric acid, reflux at 45℃ for 4h, filter, wash and dry to obtain a chromatographic separation medium. Its infrared spectrum is shown in Figure 1 , the appearance of the resin is shown in Figure 2 The infrared spectrum shows that the -1 There is infrared absorption at , which is caused by ethyl group, indicating that new groups have been successfully introduced into the resin.

[0060] Comparative Example 1

[0061] Add 300g of water, 100g of styrene, 5g of divinylbenzene, 5g of sodium chloride, 0.1g of polyvinyl alcohol, and 5g of n-butyl ether, and stir to obtain a mixture. Then add 0.2ml of 0.05% methylene blue aqueous solution to the mixture. Then add 2.0g of benzoyl peroxide, stir at a speed of 100r / min for 1h, put it into the reactor, and react at 45°C at a speed of 100r / min for 6h. After filtering, ethanol refluxes, wash and dry to obtain white spherical granular resin. Take the above 50g of resin, add 150ml of chloroform to swell for 4h, then add 100ml of 98% sulfuric acid, stir at a speed of 100r / min, and sulfonate at 70°C for 6h. After filtering, washing and drying, a yellow spherical granular resin is obtained. Its infrared spectrum is shown in Figure 3 .

[0062] Example 2

[0063] 400g water, 150g styrene, 25g divinylbenzene, 5g sodium chloride, 0.25g polyvinyl alcohol, 25g n-butyl ether were stirred to obtain a mixture. Then 0.2ml of 1% methylene blue aqueous solution was added to the mixture. Then 2.0g benzoyl peroxide was added, the speed was 250r / min, and the mixture was stirred for 2h. After loading into the reactor, the mixture was reacted for 12h at 70°C and the speed was 250r / min. After filtering, the ethanol was refluxed, and the white spherical granular resin was washed and dried. The above 50g resin was taken, 150ml chloroform was added to swell for 12h, and then 150ml of 98% sulfuric acid was added and stirred at 100r / min, and sulfonated at 85°C for 6h. After filtering, washing and drying, yellow spherical granular resin was obtained. Take 50g of sulfonated resin, add 150ml of chloroform to swell, add 200ml of sulfonyl chloride reagent, reflux at 50℃ for 8h, filter, wash with dichloromethane and dry, then add 150ml of chloroform to swell, add 100ml of dichloromethane, add 25g of α-aminoglutaric acid, reflux at 50℃ for 6h, filter, wash and dry to obtain a chromatographic separation medium. Its infrared spectrum is shown in Figure 4 .

[0064] Example 3

[0065] 500g water, 200g styrene, 50g divinylbenzene, 5g sodium chloride, 0.5g polyvinyl alcohol, 50g n-butyl ether were stirred to obtain a mixture. Then 0.2ml of 2% methylene blue aqueous solution was added to the mixture. Then 2.0g benzoyl peroxide was added, the speed was 400r / min, and the mixture was stirred for 3h. After loading into the reactor, the mixture was reacted for 18h at 95°C and the speed was 400r / min. After filtering, the ethanol was refluxed, and the white spherical granular resin was washed and dried. The above 50g resin was taken, 150ml chloroform was added to swell for 12h, and then 150ml of 98% sulfuric acid was added to sulfonate at 105°C for 6h at a speed of 100r / min. After filtering, washing and drying, yellow spherical granular resin was obtained. Take 50g of the sulfonated resin, add 150ml of chloroform to swell it, add g of sulfonyl chloride reagent and reflux at 65°C for 10h, filter, wash with dichloromethane and dry, then add 150ml of chloroform to swell it, add 100ml of dichloromethane, add 10g of α-aminoglutaric acid and reflux at 60°C for 8h, filter, wash and dry to obtain a chromatographic separation medium.

[0066] Example 4

[0067] After pretreatment, the resin of Example 1 was loaded into a pressure-resistant glass chromatography column with a size of 5*100cm. Subsequently, 500mL of a chitosan oligosaccharide mixture solution with a concentration of 600g / L was prepared and loaded at a flow rate of 0.01BV / min. After standing for 6 hours of adsorption, after reaching equilibrium, deionized water was used to wash away non-adsorbed impurities. The flow rate was 0.1BV / min. Then, 0.2‰ ammonia water was used as an eluent to elute the chitosan oligosaccharide mixture, and the effluent was collected for detection, and the single-polymerization-degree chitosan oligosaccharides were collected and combined. After testing, the purity of the separated single-polymerization-degree chitosan oligosaccharides was >95%. The TLC detection chart of the separated single-polymerization-degree chitosan oligosaccharides is shown in Figure 5 , the liquid chromatography test results are shown in Figure 6 The purity test results and the amount of one-time preparation are shown in Table 1.

[0068] Table 1 Example 4 Single degree of polymerization chitosan oligosaccharide product purity and yield

[0069]

[0070] The above examples prove that the present invention prepares a core-shell multifunctional ion exchange chromatography medium for the separation of chitosan oligosaccharides, adopts programmed temperature rise and temperature drop, synthesizes a multilayer skeleton structure, and performs step-by-step sulfonation, thereby shortening the effective mass transfer distance of the chromatography medium and shortening the chitosan oligosaccharide adsorption equilibrium time. The introduction of multifunctional groups weakens the interaction strength between chitosan oligosaccharides and the chromatography medium, improves the tailing problem existing in the chitosan oligosaccharide chromatographic separation, and has a higher separation efficiency.

[0071] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A method for preparing a chitosan oligosaccharide chromatographic separation medium, characterized in that: The steps include: (1) Dissolve styrene, divinylbenzene, sodium chloride, polyvinyl alcohol and n-butyl ether in water in a reaction kettle and stir to obtain a mixture A; (2) adding a methylene blue aqueous solution to mixture A, stirring and mixing to obtain a mixture B; (3) adding an initiator to mixture B and stirring to obtain mixture C; (4) The mixture C is placed in a reactor, and the temperature is raised and lowered under stirring conditions to react. The product is separated by filtration, and ethanol is refluxed to obtain polymer spheres D having a core-shell structure; (5) The polymer sphere D is swelled with chloroform and sulfonated to obtain a core-shell type strongly acidic ion exchange resin E; (6) Swelling the core-shell type strong acid ion exchange resin E with chloroform, adding an acyl chloride reagent for reflux reaction, washing with dichloromethane and drying to obtain a core-shell type sulfonyl chloride resin F; (7) The core-shell type sulfonyl chloride resin F is swollen with chloroform, dichloromethane is added as a reaction solvent, aminocarboxylic acid is added for reflux reaction, filtered and washed, and chitosan oligosaccharide chromatography separation medium is obtained; In step (1), in the mixture A, the mass ratio of water, styrene, divinylbenzene, sodium chloride, polyvinyl alcohol and n-butyl ether is 300-500:100-200:5-50:5:0.1:0.5:5-50; In step (3), the initiator is selected from one or more organic peroxides such as benzoyl peroxide, tert-butyl benzoyl peroxide, methyl ethyl ketone peroxide, etc.; In step (3), the mass proportion of the initiator in the mixture C after addition is 0.1-0.5%.

2. The method for preparing a chitosan oligosaccharide chromatographic separation medium according to claim 1, characterized in that: In step (2), the mass concentration of the methylene blue aqueous solution is 0.05-2%; In step (2), the relationship between the amount of the methylene blue aqueous solution added and the mixture A is 0.2 ml: 400-800 g.

3. The method for preparing a chitosan oligosaccharide chromatographic separation medium according to claim 1, characterized in that: In step (4), the temperature is programmed to rise and fall at 45-95°C with stirring for 6-18 hours.

4. The method for preparing a chitosan oligosaccharide chromatographic separation medium according to claim 1, characterized in that: In step (5), the polymer sphere D is swollen with chloroform for 4-24 hours, and the swelling volume ratio is 1:1-1:5; In step (5), the sulfonation conditions are: adding sulfuric acid for sulfonation, the volume ratio of the sulfuric acid to the solution swollen with chloroform is 1:2-1:5, and the sulfonation temperature is 70-105°C.

5. The method for preparing a chitosan oligosaccharide chromatographic separation medium according to claim 1, characterized in that: In step (6), the acyl chlorination agent is thionyl chloride, phosphorus trichloride or sulfonyl chloride; In step (6), the reflux reaction conditions are: reflux at 45-65°C for 4-10 hours; In step (7), the aminocarboxylic acid is selected from one or more of α-aminoacetic acid, α-alanine or α-aminoglutaric acid; In step (7), the reflux reaction conditions are: reflux at 45°C-60°C for 4-8h.

6. A chitosan oligosaccharide chromatographic separation medium obtained based on the preparation method according to any one of claims 1 to 5.

7. Use of the chitosan oligosaccharide chromatographic separation medium obtained by the preparation method according to any one of claims 1 to 5 in the separation and purification of chitosan oligosaccharides.

8. The use according to claim 7, characterized in that: When the chitosan oligosaccharide chromatographic separation medium is used for separating chitosan oligosaccharides with a single degree of polymerization, the method comprises the following steps: (1) The chitosan oligosaccharide chromatographic separation medium is loaded into a chromatographic column as a chromatographic filler, the chitosan oligosaccharide loading concentration is 1%-99% (w / w), and the loading amount is 0.05BV-5BV; (2) Then rinse with deionized water at 0.1-1 BV / min for 1-10 BV; (3) Finally, the chitosan oligosaccharide mixture was eluted with a mobile phase at 0-100°C at a concentration of 0.001‰-60%, and the chitosan oligosaccharides with different single polymerization degrees were combined.

Images